Analysis of the tumor suppressor gene Tsc1 in motor neuron patterning

描述（由申请人提供）：运动神经元（MNs）能够控制运动、呼吸和自主神经反应，并且受到脊髓肌萎缩症（SMA）和肌萎缩性侧索硬化症（ALS）等疾病的深刻影响。在脊椎动物中，MNs在脊髓腹侧和后脑发育，它们的细胞体沿着中枢神经系统的中外侧轴和背腹轴迁移到特定位置，它们的轴突离开中枢神经系统，以建立对肌肉目标的精确和定型的神经支配。尽管已经确定了许多在MN轴突导航中起重要作用的引导分子，但我们对控制MN轴突在特定选择点转向的信号通路的机制理解仍然是不完整的。这项工作的长期目标是确定体内控制MN轴突导航的新机制。作为一种无偏倚的方法来鉴定这些基因，使用gfp标记的MN轴突和td-番茄标记的MN细胞核的报告小鼠进行了正向遗传筛选。从ENU诱变筛选中，鉴定出9个独立突变体，并克隆出相应的基因。有趣的是，这些突变体中的一些表现出轴突靶向缺陷，其中腹侧突出的MN轴突异常地向背侧投射到感觉神经节。小鼠突变体Greenlight （GrL）中的MN寻路缺陷是由结节性硬化症复合体1 （TSC1）基因的错义突变引起的。本应用程序的目的是了解已确定的GrL/TSC1突变如何导致MN寻路缺陷。人类TSC1突变导致一系列神经表型，包括良性肿瘤、癫痫和自闭症；然而，这些症状的病理生理机制尚不清楚。核心假设是，TSC1与TSC2共同负向调控ephrin/Eph下游mTOR通路。这一假设是基于我筛选的GrL/TSC1突变的鉴定，以及最近对抑制性轴突引导信号负调控mTOR通路的观察而提出的。提出这项研究的基本原理是，对TSC1功能的详细了解有可能转化为对结节性硬化症患者的改进治疗干预。在强有力的初步数据指导下，我们将通过两个具体目标来验证这一假设：1)研究GrL/TSC1突变如何影响TSC1/TSC2复合物的形成，并详细分析GrL/TSC1突变如何影响发育中的PNS中的MN和感觉轴突模式。2)研究ephrin/EphA是否在TSC1上游起作用，以及TSC1下游TOR复合物1调控的改变是否导致GrL MN寻路缺陷。提出的实验是创新的，因为它们是基于GrL/TSC1等位基因作为一种导致MN寻路缺陷的新突变的识别。最终，所获得的知识有可能告知肿瘤抑制基因TSC1如何在神经系统发育中起作用，以及该途径的扰动如何导致TSC患者典型的神经系统表现。

DESCRIPTION (provided by applicant): Motor neurons (MNs) enable control over locomotion, respiration and autonomic responses, and are profoundly affected by diseases such as spinal muscle atrophy (SMA) and amyotrophic lateral sclerosis (ALS). In vertebrates, MNs develop in the ventral spinal cord and hindbrain, their cell bodies migrate to specific positions along the medio-lateral and dorso-ventral axes of the CNS and their axons exit the CNS to established precise and stereotypic innervation of muscle targets. Although numerous guidance molecules have been identified that play an essential role in MN axon navigation, our mechanistic understanding of the signaling pathways that control MN axon steering at specific choice points remains fragmentary. The long-term goal of the proposed work is to identify novel mechanisms that control MN axon navigation in vivo. As an unbiased approach to identify such genes, a forward genetic screen was carried out using a reporter mouse with GFP-labeled MN axons and td-tomato-labeled MN nuclei. From the ENU mutagenesis screen, nine independent mutants were identified and their corresponding genes were cloned. Interestingly, several of these mutants display axon targeting defects in which ventral-projecting MN axons aberrantly project dorsally into the sensory ganglia. The MN pathfinding defects in a mouse mutant called Greenlight (GrL) are caused by a missense mutation in the tuberous sclerosis complex 1 (TSC1) gene. The objective in this application is to understand how the identified GrL/TSC1 mutation contributes to MN pathfinding defects. Mutations in human TSC1 cause a spectrum of neurological phenotypes including benign tumors, epilepsy and autism; however the pathophysiology of these symptoms is poorly understood. The central hypothesis is that TSC1, in conjunction with TSC2, negatively regulates the mTOR pathway downstream of ephrin/Eph in developing MNs. This hypothesis has been developed based on the identification of the GrL/TSC1 mutation in my screen, and the recent observation that inhibitory axon guidance cues negatively regulate the mTOR pathway. The rationale for the proposed research is that a detailed understanding of TSC1 function has the potential to translate into improved therapeutic interventions in patients suffering from tuberous sclerosis. Guided by strong preliminary data, the hypothesis will be tested by pursuing two specific aims: 1) to study how the identified mutation in GrL/TSC1 influences TSC1/TSC2 complex formation and to analyze in detail how the GrL/TSC1 mutation affects MN and sensory axon patterning in the developing PNS. 2) to study whether ephrin/EphA operates upstream of TSC1 and whether altered regulation of the TOR complex 1 downstream of TSC1 results in the GrL MN pathfinding defects. The proposed experiments are innovative because they are based on the identification of the GrL/TSC1 allele as a novel mutation that causes MN pathfinding defects. Ultimately, the knowledge gained has the potential to inform how the tumor suppressor gene TSC1 functions in nervous system development and how perturbation of this pathway can cause the neurological manifestations typically observed in TSC patients.